Heart failure commonly results from ischemic or hypertensive disease, and novel strategies are sorely needed to address the ever-expanding population of patients with heart failure world-wide. Over the past two years we have explored the role of a novel cardiac-specific protein kinase, troponin I type 3 interacting kinase (gene name TNNI3K) in regulating ischemic injury, post-MI remodeling, and pressure overload-induced remodeling. We have employed a number of strategies including 1) transgenic mice expressing either wild-type, constitutively active, or kinase-inactive TNNI3K, 2) conditional cardiac-specific deletion of TNNI3K (CKO mice), and 3) novel small molecule inhibitors of TNNI3K generated by our collaborators at Glaxo-Smith-Kline that are able to be used both in vitro and in vivo. All of our data support two key conclusions: 1) Inhibition of TNNI3K is strongly protective, and activation of TNNI3K is highly detrimental, in the setting of ischemia/reperfusion (I/R) injury, and 2) TNNI3K appears to be a central regulator of fibrotic remodeling in the heart. We now propose to determine the molecular mechanisms by which TNNI3K regulates these processes, thereby expanding our understanding of mechanisms of ischemic injury and repair. In our Specific Aims we propose to identify TNNI3K targets from genomic analyses and have already identified a profound role of TNNI3K in regulating expression of genes implicated in fibrotic remodeling. This is consistent with the significant reduction in fibrosis we observe in mice subjected to thoracic aortic constriction that were treated with one of the small molecule inhibitors. We will also employ proteomic approaches to identify direct substrates of TNNI3K using the analog sensitive kinase allele (ASKA) approach pioneered by Shokat and co-workers. Finally, we will focus on the role of select substrates identified in Aim 2, and will determine ther role in ischemic injury and post-infarct remodeling. Given our successful use of small molecule inhibitors targeting TNNI3K in vivo, and the demonstrated safety of this approach in our pre-clinical models, we believe that the stage could be set for fairly rapid translation to clinical trals if our hypotheses are borne out.